Anti-lymphotoxin-beta receptor antibodies as anti-tumor agents
a technology of anti-lymphotoxin and beta receptor, which is applied in the direction of antibody medical ingredients, tumor necrosis factor, peptide/protein ingredients, etc., can solve the problems of general toxicity in humans and preclude its therapeutic application in humans, and achieve the effect of increasing the percentage of tumor cells killed
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example 1
Generation of Baculovirus-Infected Insect Cell Supernatants Containing LT-α / β Forms
[0134] Recombinant baculovirus encoding either full length LT-α or a secreted form of LT-β were made as described (Crowe et al., Science, 264, pp. 707-710 (1994)). High five insect cells (Invitrogen, San Diego, Calif.) were inoculated at a density of 2×105 cells / ml into 7.2 liters of SF 900-II (Gibco) media without serum. The culture reached 1.8×106 cells / ml 48 hours later and was infected with 150 ml (3×108 PFU / ml) of LT-β and 300 ml of LT-α baculovirus stocks. Two days later, the culture was harvested and the cell debris was removed by centrifugation. After addition of EDTA and PMSF (1 mM EDTA and 150 μM PMSF final concentration), the clarified supernatant was concentrated 10-fold by ultrafiltration using a S1YM10 (Amicon) spiral cartridge. The concentrate was divided into six 120 ml portions and aliquots were stored at −70 C prior to purification.
example 2
Preparation of Soluble LT-β Receptors as Immunoglobulin Fc Chimera
[0135] The extracellular domain of LT-β-R up to the transmembrane region was amplified by PCR from a cDNA clone using primers that incorporated NotI and SalI restriction enzyme sites on the 5′ and 3′ ends, respectively (Browning et al., J. Immunol., 154, pp. 33-0.46 (1995)). The amplified product was cut with NotI and SalI, purified and ligated into NotI-linearized vector pMDR901 along with a SalI-NotI fragment encoding the Fc region of human IgG1. The resultant vector contained the dihydrofolate reductase gene and the LT-β-R-Fc chimera driven by separate promoters. The vector was electroporated into CHO dhfr− cells and methotrexate-resistant clones were isolated as per standard procedures. The LT-β-R-Fc is secreted into the medium and an ELISA assay was used to select for cell lines producing the highest level of the chimeric protein. A high-producing cell line was grown to large numbers and conditioned medium colle...
example 3
Affinity Purification of LT-α1 / β2 Using TNF-R and LT-β-R
[0136] To prepare resins for the receptor affinity purification of LT forms, purified preparations of LT-β-R-Fc (as described in Example 2 herein) and TNF-R p60-Fc (Crowe et al., Science, 264, pp. 707-10 (1994)) were immobilized on CNBr-sepharose (Pharmacia) at 5 mg / ml resin essentially following the manufacturer's specifications. The resins were put through one elution cycle prior to use. A portion (120 ml) of the S1Y10 concentrate was passed over two sequential p60 TNF-R-Fc columns, which bind LT-α and LT-α2 / β1. The flow through, which contained LT-α1 / β2 and LT-β, was passed over a LT-β-R-Fc column. The column was washed with 5 volumes each of PBS, PBS with 0.5 M NaCl and PBS, and then the LT-α and LT-α2 / β1 complexes were eluted with 25 mM sodium phosphate, 100 mM NaCl, pH 3.5. Elution fractions were immediately neutralized with 1 / 20 volume of 0.5 M sodium phosphate, pH 8.6 and stored on ice. Fractions containing protein wer...
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